Methods for treating viral infections

ABSTRACT

The present invention provides a method for treating a viral infection in a subject in need of such treatment comprising administering to the subject a photosensitizer formulated in a liposome carrier, and exposing the subject to light at a wavelength 20-40 nm greater than the maximum absorption of the photosensitizer at a sufficient dose and duration to treat the viral infection in the subject. The present invention also provides a method for treating a viral infection in a subject in need of such treatment comprising administering to the subject (i) a photosensitizer formulated in a liposome carrier and (ii) at least one quencher, and exposing the subject to light at a sufficient wavelength, dose and duration to treat the viral infection in the subject.

This is a continuation of application Ser. No. 08/841,042, filed Apr.29, 1997, now U.S. Pat. No. 6,103,706 the contents of which is herebyincorporated by reference.

BACKGROUND OF THE INVENTION

Current treatments for AIDS use drugs; such as azidothymidine (AZT), andother protease inhibitors which inhibit HIV replication. Thesetreatments have only limited efficacy because HIV mutates rapidly andnew strains that are drug-resistant eventually develop.

The use of photochemical inactivation procedures are currently used torender blood safe for transfusion. For example, the addition ofphthalocyanines such as Pc4 to blood followed by exposure to red lighthas been shown to effectively reduce viral load in vitro(Margolis-Nunno, H., et al. Transfusion 36:743-750 (1996)). Pc4 killsHIV by binding to the viral envelope and producing reacting oxygenspecies (ROS) upon light activation, which subsequently cause damage tothe viral proteins, rendering the virus non-infectious. Because bindingof Pc4 to viral membrane is nonspecific, drug-resistant strains cannotarise from this treatment.

Accordingly, if such treatment could be performed on AIDS patients toreduce HIV viremia in their blood, such treatment is expected to bebeneficial since a reduced quantity of HIV in plasma is a predictor forenhanced survival of AIDS patients (Mellors, J. W., et al. Science272:1167-1170 (1996)). However, because photo-inactivation is notspecific, Pc4 and other photosensitizers, can also bind to red bloodcell (RBC) membranes and cause RBC damage. Accordingly, there exists aneed for rendering RBC safe while at the same time maintaining goodviral kill in vivo.

SUMMARY OF THE INVENTION

The present invention is directed to a method for treating a viralinfection, such as HIV, in a subject in need of such treatment, whichutilizes photochemical inactivation procedures to reduce the level ofinfectious virus contained in the blood while simultaneously minimizingthe damage done to red blood cells. In this connection, the inventor hasfound that by incorporating the photosensitizer in a liposomeformulation and applying light at a certain wavelength above the maximumabsorption of the photosensitizer, a good virus kill can be achievedwhile minimizing the damage to red blood cells.

In addition, the inventor believes that by incorporating thephotosensitizer in a liposome formulation and also administering aquencher alone or in a liposome formulation, the damage to red bloodcells also can be minimized.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts VSV inactivation in whole blood (spiked with 6.1 log₁₀VSV; ◯) and plasma (spiked with 6.3 log₁₀ VSV; ) following treatmentwith liposomal 2 μM Pc4 and liposomal 2 μM Pc4+4 mM GSH+5 mM trolox,respectively, and 700 nm light at various light fluences (J/cm²) at afluence rate of 20 mW/cm² at room temperature.

FIG. 2 depicts the extent of hemolysis of red blood cells in storagefollowing treatment with liposomal 2 μM Pc4 and 700 nm light at 6 J/cm²(□) or 7.5 J/cm² (▪), at a fluence rate of 20 mW/cm² at roomtemperature. Untreated RBC was the control (◯).

FIG. 3 depicts HIV inactivation in whole blood (spiked with >5.6 log₁₀HIV; ◯) following treatment with liposomal 2 μM Pc4 and 700 nm light atvarious light fluences (J/cm²) at a fluence rate of 20 mW/cm² at roomtemperature.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for treating a viral infectionin a subject in need of such treatment. The method comprisesadministering to the subject a photosensitizer formulated in a liposomecarrier, and exposing the subject to light at a wavelength 20-40 nmgreater than the maximum absorption of the photosensitizer at asufficient dose and duration to treat the viral infection in thesubject.

The method of the present invention may be used to treat various viralinfections caused by viruses including but not limited to humanimmunodeficiency virus, Cytomegalovirus, Ebstein-Barr virus, Hepatitis Bvirus, Hepatitis C virus, Herpes Simplex type I and II viruses, andother viruses that circulate in freely in blood, as well ascell-associated viruses. As used herein, “treating” means that the levelof infectious virus is reduced in such a manner that the majority of allof the infectious virus contained in the blood of the subject isdestroyed or inactivated. The “subject” may be a human or an animalsubject, and is preferably a human subject.

Suitable photosensitizers include but are not limited tophthalocyanines, porphyrins, purpurins, psoralens, bergaptens,angelicins, chlorins and flavins. Particularly preferredphotosensitizers are those compounds which absorb in the red region ofthe visible spectrum such as phthalocyanines. Suitable phthalocyaninesinclude but are not limited to phthalocyanines containing a central atomof aluminum, germanium, gallium, tin or silicon such as siliconphthalocyanine (i.e. hydroxysiloxydimethylpropyl-N-dimethyl siliconphthalocyanine, “Pc4”)), as well as sulfonated or nitrated forms of suchpthalocyanines, such as sulfonated aluminum phthalocyanine (i.e.aluminum tetrasulfophthalocyanine (“AlPcS₄”) or aluminumdisulfophthalocyanine (“AlPcS_(2a)”). Such phthalocyanines and othersare described in Spikes, J. Photochemistry and Photobiology 43:691-699(1986); Ben-Hur, E. and Rosenthal, I. Int. J. Radiat. Biol. 47:145-147(1985); Moser, F. H. and Thomas, A. C. The Phthalocyanines, Boca Raton,CRC Press, 1984; Kreimer-Birnbaum, M. Sem. Hematol. 26:157-193 (1989);and U.S. Pat. Nos. 5,120,649, 5,232,844 and 5,484,778, which are herebyincorporated by reference in their entirety. In the most preferredembodiment, the phthalocyanine is Pc4.

The photosensitizer may be formulated in the liposome carrier by mixingthe desired amount of the photosensitizer with the liposome carrierusing procedures well known in the prior art. The liposome carrier maycomprise at least one natural phospholipid (e.g. soy phosphatidylcholine), at least one synthetic phospholipid, or combinations thereof.Suitable synthetic liposome carriers include but are not limited to oneor more of the following: 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine(POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS),1,2-dioleolyl-sn-glycero-3-phosphate (PA),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt(DSPG), and 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC).Preferably, the liposome carrier comprises POPC and DOPS at a ratio of10:1-0.5:1. Most preferably, POPC and DOPS are used at a ratio of about4:1, since the inventor has found that this ratio results in the leastamount of damage to red blood cells. These and other liposomeformulations are discussed in application Ser. No. 08/841,042, filedApr. 29, 1997, entitled “Methods for Viral Inactivation and Compositionsfor Use in Same”, which is hereby incorporated by reference.

The amount of the photosensitizer administered in thephotosensitizer/liposome formulation will depend upon thephotosensitizer chosen. However, when the photosensitizer is Pc4, theamount administered is preferably 0.3-3.0 mg/kg body weight, and mostpreferably is about 1 mg/kg body weight of the subject. Thephotosensitizer/liposome formulation may be administered by methodsknown to those skilled in the art, and preferably by transfusion orinjection. For such modes of administration, thephotosensitizer/liposome formulation may be combined with apharmaceutically acceptable carrier which is “acceptable” in the senseof being compatible with the other ingredients of the formulation andnot deleterious to the recipient thereof. For example, thephotosensitizer/liposome formulation may be combined with a sterileaqueous solution which is preferably isotonic with the blood of therecipient. Such formulations may be prepared by combining thephotosensitizer/liposome formulation with water containingphysiologically compatible substances such as sodium chloride, glycine,and the like, and having a buffered pH compatible with physiologicalconditions to produce an aqueous solution, and rendering said solutionsterile. The formulations may be present in unit or multi-dosecontainers such as sealed ampoules or vials.

The light is applied about 10 minutes to about 3 hours, and preferablyabout 15-60 minutes, after administration of the photosensitizer. Thelight should be applied at a sufficient wavelength, dose and duration tomaximize the inactivation of infectious virus, and at the same time, tominimize the damage to the red blood cells. The inventors have foundthat if the wavelength of light applied is 20-40 nm greater than themaximum absorption of the photosensitizer, this should be sufficient tomaximize the inactivation of infectious virus while minimizing thedamage to the red blood cells. For example, when the photosensitizer isPc4, it is preferred that the light be applied at a specific wavelengthsomewhere in the range of 695-705 nm, and most preferably at a specificwavelength of about 700 nm.

The specific dose and duration of light again will depend upon thephotosensitizer chosen. When the Pc4 is the photosensitizer, the dose oflight applied is preferably about 5-25 mW/cm², and most preferably about18-22 mW/cm², while the duration of light application is about 5-60minutes, and most preferably about 20-30 minutes. Suitable sources oflight include commercially available lasers, lamps, light emittingdiodes and the like. Preferably, a LED arrays (Efos Canada, Inc.,Mississauga, Ontario, Canada) is employed. To achieve the desiredwavelength of light, the lamp may be equipped with commerciallyavailable filters.

It also is within the confines of the present invention that one or morequenchers can be administered before, during or after the administrationof the photosensitizer/liposome formulation, but before application oflight. Suitable quenchers include but are not limited to glutathione,trolox, flavonoids, vitamin C, vitamin E, cysteine and ergothioneine andother non-toxic quenchers, and preferably vitamin E. The amount of thequencher administered will depend upon the specific quencher(s) chosenand can be determined by one skilled in the art. However, when thequencher is vitamin E, the preferred dose ranges from about 10 mg/kgbody weight to about 1 g/kg body weight, and most preferably about 100mg/kg body weight.

In addition, it is within the confines of the present invention that oneor more quenchers can be formulated in a liposome carrier to enhance theassociation of the quencher to the red blood cells, thus affording amore selective protection to the red blood cells. Suitable liposomecarriers include those carriers that enhance delivery of the quencher tothe red blood cells, such as liposomes containing cholesterol, liposomesmade from natural phospholipids (e.g. soy phosphatidyl choline (PC)),and POPC. Preferably, the quencher is vitamin E, and the liposomecarrier is POPC. When using vitamin E and POPC, the preferred vitaminE:POPC ratio is 1:5-1:3, and most preferably is about 1:3.7.

The present invention is described in the following Examples which areset forth to aid in an understanding of the invention, and should not beconstrued to limit in any way the invention as defined in the claimswhich follow thereafter.

EXAMPLE 1 Preparation of Pc4 in Liposomes

HOSiPcOSi(CH₃)₂(CH₂)₃N(CH₃)₂ (Pc4) (1 mg) was dissolved in 0.5 ml ofN-methylpyrolidone (NMP) prewarmed to 50° C. and sonicated 1-2 minutes.1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) (90 mg) wasdissolved in tert-butyl alcohol (0.5 ml), and prewarmed to 50° C.Similarly, 1, 2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) (10 mg)was dissolved in tert-butyl alcohol (0.5 ml), and prewarmed to 50° C.The solutions were then sonicated at 50° C. until complete dissolutionoccurred. The phospholipid solutions were combined and kept at 50° C.The Pc4 solution was combined with the phospholipid solution at 50° C.to achieve a ratio of Pc4:phospholipid=1:100 (w/w). This was mixed withexcess (16×) of aqueous solution containing 9.45% D-lactose and 0.027%NaCl. Prior to mixing, the organic solution should be at 50° C. and theaqueous solution at 4° C. The former was added dropwise into the latterwith vigorous stirring. Stirring was continued for 15 minutes at 4° C.The resulting liposomal suspension was concentrated (10×) with 100 kDaAmicon concentrator and centrifuged at 3000 rpm at room temperature. Theremaining organic solvents were removed by dialysis against lactose/NaClsolution at 4° C. using a dialysis membrane with a MW cutoff of6000-8000. The dialysis solution was changed 3× every few hours. Thefinal liposomal solution of Pc4 was lyophilized and stored at 4° C.Prior to use, the liposomes were dissolved in phosphate buffered saline(PBS) using a vortex for mixing followed by 1-2 minutes of sonication.

EXAMPLE 2 VSV In Vitro Study

The inactivation kinetics of vesicular stomatitis virus (VSV) in wholeblood and plasma with liposomal Pc4 and 700 nm light was studied. Thesekinetics are identical to that of HIV (Margolis-Nunno, H., et al.Transfusion 36:743-750 (1996)). Whole blood and plasma samples werespiked with 6.3 and 6.1 log₁₀ of VSV, respectively. The whole bloodsample was then treated with the Pc4 liposome composition prepared inExample 1 to a Pc4 concentration of 2 μM. The plasma sample was treatedwith the Pc4 liposome to a Pc4 concentration of 2 μM, and also with 4 mMGSH and 5 mM trolox. Using an LED array (Efos Canada, Inc., Mississauga,Ontario, Canada) emitting at 700 nm, light was applied to the wholeblood and plasma samples at various light fluences (J/cm²) at a fluencerate of 20 mW/cm². The results are presented in FIG. 1. The majority ofthe VSV contained in the plasma was killed at light fluences greaterthan 2 J/cm², while similar virus kill in the whole blood was obtainedwith a fluence greater than 6 J/cm².

EXAMPLE 3 Evaluation of RBC Damage

Whole blood was treated with the Pc4 liposome composition prepared inExample 1 to a Pc4 concentration of 2 μM. 700 nm light was then appliedat doses that inactivate ≧6 log₁₀ VSV (6 and 7.5 J cm²), and stored at4° C. The extent of RBC hemolysis during storage was determined bycomparing the hemoglobin in the supernatant to the total hemoglobin. Thetotal hemoglobin was determined by using the Drabkin reagent (SigmaProcedure #525, Sigma Chemical Co., St. Louis, Mo.). The absorption at540 nm was used to calculate the amount of hemoglobin released in thesupernatant. The results are presented in FIG. 2 and show no significanthemolysis of the treated RBC over that of control, untreated RBC.

EXAMPLE 4 HIV In Vitro Study

Whole blood spiked with >5 log₁₀ HIV was treated with the Pc4 liposomecomposition prepared in Example 1 to a Pc4 concentration of 2 μM. Usingan LED array emitting at 700 nm, light was applied to the whole blood atvarious light fluences (J/cm²) at a fluence rate of 20 mW/cm². HIV titerwas assayed as described (Margolis-Nunno, H., et al. Transfusion36:743-750 (1996)). The results are presented in FIG. 3, and show asignificant reduction of HIV titers at a light fluence of 8 J/cm².

EXAMPLE 5 The Use of Quenchers In Vitro

RBCC were treated with 2 μM Pc4 in POPC:DOPC=4:1 liposomes in thepresence of 1 mM tocopherol succinate in the formulations presented inTable 1 below, and exposed to 10 J/cm² of red light (670 nm) emitted byLED array. The RBCC were then stored for 21 days at 4° C. The percentageof hemolysis for each formulation is presented in Table 1 below.

TABLE 1 Formulation of α-tocopherol Hemolysis After succinate (1 nM) 21Day Storage (%) Ethanol 10.5 POPC:DOPS = 4:1 18.3 POPC:DOPS:chol =9:1:10 6.1 POPC 1.2 Untreated RBCC 1.1

EXAMPLE 6 In Vivo Study

Mice were administered liposomal Pc4 at 1 mg/kg body weight, their backswere shaved and then exposed to 700 nm light at 20 mW/cm², 15 minutesafter Pc4 administration. The mice tolerated this treatment for up to 30minutes illumination with no adverse effects. This treatment is expectedto cause sufficient exposure of the circulating blood to reduce viremiain plasma by ≧6 log₁₀.

All publications and patents mentioned hereinabove are herebyincorporated by reference in their entirety. While the foregoinginvention has been described in some detail for purposes of clarity andunderstanding, it will be appreciated by one skilled in the art from areading of the disclosure that various changes in form and detail can bemade without departing from the true scope of the invention in theappended claims.

What is claimed:
 1. A method for treating a viral infection in a subjectin need of such treatment comprising administering to the blood of thesubject (i) a photosensitizer formulated in a first liposome carrier and(ii) at least one quencher, and exposing the subject to light at asufficient wavelength, dose and duration to treat the viral infection inthe subject; said first liposome carrier comprising1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) and1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS) at a ratio of10:1-0.5:1.
 2. The method of claim 1, wherein the viral infection iscaused by a virus selected from the group consisting of humanimmunodeficiency virus, Cytomegalovirus, Ebstein-Barr virus, Hepatitis Bvirus, Hepatitis C virus, and Herpes Simplex viruses.
 3. The method ofclaim 2, wherein the virus is human immunodeficiency virus.
 4. Themethod of claim 1, wherein the photosensitizer is selected from thegroup consisting of a phthalocyanine, a porphyrin, a purpurin, apsoralen, a bergapten, an angelicin, a chlorin, and a flavin.
 5. Themethod of claim 4, wherein the photosensitizer is a phthalocyanine. 6.The method of claim 5, wherein the phthalocyanine is an aluminum,germanium, gallium, tin or silicon phthalocyanine; asulfonated-aluminum, germanium, gallium, tin or silicon phthalocyanine;or a nitrated-aluminum, germanium, gallium, tin or siliconphthalocyanine.
 7. The method of claim 6, wherein the phthalocyanine isa sulfonated aluminum phthalocyanine.
 8. The method of claim 7, whereinthe sulfonated aluminum phthalocyanine is aluminumtetrasulfophthalocyanine or aluminum disulfophthalocyanine.
 9. Themethod of claim 6, wherein the phthalocyanine is a siliconphthalocyanine.
 10. The method of claim 9, wherein the siliconphthalocyanine is hydroxysiloxydimethylpropyl-N-dimethyl siliconphthalocyanine (Pc4).
 11. The method of claim 1, wherein the ratio ofPOPC to DOPS is about 4:1.
 12. The method of claim 1, wherein thephthalocyanine formulated in the first liposome carrier is administeredby transfusion or injection.
 13. The method of claim 1, wherein thequencher is selected from the group consisting of flavonoids, vitamin C,vitamin E, glutathione, trolox, cysteine, ergothioneine, and othernon-toxic quenchers.
 14. The method of claim 13, wherein the quencher isformulated in a second liposome carrier.
 15. The method of claim 14,wherein the second liposome carrier comprises at least one naturalphospholipid, at least one synthetic phospholipid, or combinationsthereof.
 16. The method of claim 15, wherein the second liposome carriercomprises cholesterol.
 17. The method of claim 15, wherein the secondliposome carrier comprises soy phosphatidyl choline (PC).
 18. The methodof claim 15, wherein the second liposome carrier comprises1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC).
 19. The method ofclaim 18, wherein the quencher is vitamin E.
 20. The method of claim 19,wherein the ratio of vitamin E to POPC is 1:5-1:3.
 21. The method ofclaim 19, wherein the ratio of vitamin E to POPC is about 1:3.7.
 22. Themethod of claim 1, wherein the quencher is administered before, duringor after administration of the photosensitizer formulated in the firstliposome carrier.
 23. The method of claim 1, wherein the light isapplied at a wavelength 20-40 nm greater than the maximum absorption ofthe photosensitizer.
 24. The method of claim 1, wherein the light isapplied about 10 minutes to about 3 hours after administering thephotosensitizer formulated in the first liposome carrier.
 25. The methodof claim 1, wherein the light is applied about 15-60 minutes afteradministering the photosensitizer formulated in the first liposomecarrier.
 26. The method of claim 10, wherein the amount of Pc4administered is about 0.3-3.0 mg/kg body weight of the subject.
 27. Themethod of claim 10, wherein the amount of Pc4 administered is about 1mg/kg body weight.
 28. The method of claim 10, wherein the wavelength oflight applied is about 695-705 nm.
 29. The method of claim 10, whereinthe wavelength of light applied is about 700 nm.
 30. The method of claim10, wherein the dose of light applied is about 5-25 mW/cm².
 31. Themethod of claim 10, wherein the dose of red light is about 18-22 mW/cm².32. The method of claim 10, wherein the light is applied for about 5-60minutes.
 33. The method of claim 10, wherein the light is applied forabout 20-30 minutes.